The inner membrane of the mitochondria has several embedded transport proteins that facilitate the translocation of metabolites and cofactors into the mitochondria. These proteins work in concert with mechanisms in the outer membrane to bring cationic and anionic metabolic intermediates from the cytosol to the matrix of mammalian cells. There are 35 inner mitochondrial wall transporters recognizable in the yeast genome; the function of only 13 are known. We have recently cloned a cDNA that encodes an inner mitochondrial protein that facilitates the transport of folate cofactors into the mitochondria. There are data suggesting that a component of the toxicity of the tetrahydrofolate antimetabolites seen in man may be due to inhibition of this transporter. Mammalian cells deficient in this transporter cannot survive in the absence of glycine. In this application, we propose to study the mechanism of this transporter, defining the substrate specificity for the various forms of the folates found in cells and also for the various antifolates that have been used to treat human cancers. Transport process will be studied in isolated mitochondria and in recombinant protein reconstituted into proteolipid bilayers. The proteins in the outer and inner mitochondrial membrane that bind folates will be defined, as will any binding partners involved in the transport through inner or outer membrane of the mitochondria. The phenotype of mice genetically engineered to lack this transporter will be studied as a model for human genetic deficiencies of mitochondrial folate transport. The binding site for folates in this transporter and the orientation of the transmembrane domains will be determined by site-directed mutagenesis, random mutagenesis of peptides, and epitope mapping. These studies will lead to an understanding of the basic biochemistry of this transport process and will lead to an understanding of the role of the inner membrane folate transporter in antifolate toxicity and in human genetic disorders of folate metabolism.